NKT Cells, the CD1d System and Glycolipids from Marine Sponges, Bacterial Cells, and Human Tissues (1).
NKT cells are defined by the expression of a semi-invariant, CD1d-restricted, alpha-beta T cell receptor (TCR). Most of these receptors are Vα14-Jα18/Vβ11 in structure. Human Vα24 NKT cells bind and react strongly to the CD1d receptor on dendritic cells, once this receptor is bound to certain glycolipids like alpha-galactosylceramide, the chemical structure of which is shown in FIG. 1A (originally isolated from the marine sponge: Agelas mauritianus which was first collected from the Okinawan Sea), and glycolipids isolated from the cell wall of Sphingomonas (the chemical structures of which are presented in FIG. 1B) which is a Gram-negative, LPS-negative member of the alpha-proteobacteria class (1). A mixture of the CD1d receptor mixed with either alpha-galactosylceramide, or the glycolipids shown in FIG. 1B from Sphingomonas (both of which are comprised of a sugar moiety linked to the ceramide lipid by an alpha glycolipid linkage (1) will induce activation of NKT cells through binding to the Vα14-Jα18/Vβ11 receptor on human Vα24 NKT cells (1). Experimental testing shows that the injection of such glycolipids into mouse tumor models induce tumor regressions and extends survival of tumor bearing mice (1). It is further noted that the glycospingolipids once bound to the CD1d receptor on dendritic cells are responsible for the strong stimulation of NKT cells and their role in clearing infections (1).
Mixtures of CD1d with the glycosphingolipid iGb3 (the chemical structure of which is presented in FIG. 1C, which is found in mouse and human tissues (1), induces activation of human NKT cells, also through binding of the Vα14-Jα18/Vβ11 receptor on human Vα24 NKT cells (1). The level on stimulation by iGb3 shown in FIG. 1C, in which a sugar moiety is linked to ceramide through a beta linkage, is much weaker that that seen with the glycolipids shown in FIGS. 1A and 1B, in which the sugar moiety is linked to the ceramide through an alpha linkage (1).
The CD1d receptor is a 37.713 kilodaltons (kDa) protein with 333 amino acids: 18 amino acids in the signal sequence, 282 amino acids in the extracellular domain, 23 amino acids in the transmembrane domain and 10 amino acids in the cytoplasmic domain (2). The aminoacid structure of the extracellular domain of the CD1d receptor (SEQ ID No. 1) is shown in FIG. 1D which is preferable although use of the entire CD1d protein (333 aminoacids) is acceptable. Both in vitro and in vivo experiments show that once any one of the glycolipids shown in FIGS. 1A-1C are mixed with the entire CD1d receptor or ecdCD1d receptor (FIG. 1D), the complex thus formed displays a high affinity to the Vα14-Jα18/Vβ11 receptor on human Vα24 NKT cells.
Non Vα24 NKT cells which are CD1d restricted may be involved in autoimmune diseases (1). The CD1d system of MHC like molecules on dendritic cells (DCs) are thought to present lipid antigens to T cells. The combination of alpha-galactosylceramide like glycolipids (AGCLGL) with the CD1d on DCs binds to the mouse TCR of the NKT cells with a dissociation constant (Kd) of 100 nM and with a Kd of 7 μM to the human TCR (1). This results in the activation of expression of ligands such as CD40L on the NKTs, and the release from the NKT cells of Th1 and Th2 cytokines and chemokines (see FIGS. 2A-2D). As a consequence of a bacterial infection these cytokines are released from the activated NKT cells along with the binding of the CD40L of the activated NKT cells to the CD40 receptor on the DCs and results in their activation (see FIGS. 2A-2D).
CD1d and MHC Presentation Molecules on DCs.
DCs become activated through binding of external ligands to plasma membrane receptors (e.g. the CD40 ligand/CD40 receptor) so as to increase expression of Class I and Class II MHC for the presentation of peptide fragments of target protein antigens to CD8 effector T cells (see FIG. 3) and to increase the level of expression of surface molecules like CD1d for presentation of lipid antigens like, for example, alpha-galactosylceramide (See FIGS. 2A-2D).
CD1d is expressed on DCs, cortical thymocytes, as well as B cells. CD1d is also found on hepatocytes in virally infected livers, as well as glial cells from inflamed tissues. CD1d is not found on any other cells except at very low levels.
Bacterial Glycolipids and NKT Cells.
Alpha-galactosyldiacylglycerols extracted from Gram-negative LPS negative organisms (such as Borrelia burgdorferi which causes Lyme disease) can directly stimulate NKT cells. Most of the evidence indicates that NKT cells and their hVα24-Jα18 TCRs have the function of binding and recognizing α-galactosylceramide (AGC) like glycolipid (AGCLGL) ligands from bacterial cells so as trigger an innate like immune response (1) as well as an adaptive immune response.
AGC was first isolated from the marine sponge Agelas mauritanitus. It was shown that when AGC binds to the CD1d receptor on DCs, it can bind to the invariant antigen recognition receptor (IARR) of NKT cells and activate them. AGCLCL antigens have been isolated from the following infectious agents which have been shown to bind CD1d resulting in the subsequent binding of the ACLGL-CD1d combination to the IARR of NKT: (i) monoglycosylcderamides from Spongomonas species, (ii) phosphatidylinositol mannosides from Mycobacterium tuberculosis, (iii) lipophosphoglycan from Leishmania donovani. These AGCLGL molecules presumably have similarities in structure to AGC. Applicant submits that all infectious antigens, foreign antigens and/or self-antigens, of any kind or character, that carry glycolipid molecules that are similar in structure, fall within the confines of Applicant's invention.
Mechanism of the Immune Response to Invading Bacterial Cells Positive for α-Galactosylceramide Like Glycolipid Ligands.
The invasion of a microbe positive for glycolipids similar to α-galactosylceramide leads to the binding of the α-galactosylceramide like antigens (AGCLGL) to the CD1d molecule expressed on resting DCs. The formation of the α-galactosylceramide-CD1d or AGCLGL-CD1d combination creates a structure which has a high binding affinity for the hVα24-Jα18 TCRs of NKT cells.
Binding of Glycolipids to CD1d on DCs Leads to Activation of the CD40L/CD40 Receptor Pathway.
The binding of the α-galactosylceramide-CD1d or AGCLGL-CD1d combination to the hVα24-Jα18 TCRs leads to activation of the NKT cells, with consequent increase of the level of the immunostimulatory molecule, CD40 ligand (CD40L), on the surface of the NKT cell (see FIGS. 2A-2D). In addition, the binding triggers the release from the NKT cells of large amounts of Th1 like cytokines (interferon-γ, IL-12, and interferon-α), Th2 like cytokines (IL-4), and increased expression of the B7.1 and B7.2 co-stimulatory molecules (1).
Interaction of NKT Cells and DCs.
The expression of the CD40L on the NKT cells then leads to binding of the CD40L on the NKT cell surface to the CD40 receptor on the DCs (see FIG. 2), the very same DCs which have the α-galactosylceramide bound to their own CD1d receptor. These α-galactosylceramide-CD1d combinations on the DC bind to the hVα24-Jα18 TCRs on the NKT cells. When the DC becomes activated by the CD40L of the activated NKT, then these DCs migrate to the draining lymph nodes where they present their α-galactosylceramide-CD1d or AGCLGL-CD1d combinations as well as their TAA to appropriate T and B cells to induce an adaptive immune response to the bacterial cell glycolipids and TAA.
Historical Summary of the Development of the TAA/ecdCD40L Vaccine Platform the Development of which the Applicant Participated as a Co-Inventor.
Previous Vaccines.
Vaccines have been described that include an adenoviral expression vector encoding a fusion protein that includes a target associated antigen (TAA) fused to the CD40 ligand (CD40L). See, e.g., U.S. Patent Application Publication US 2005-0226888 (application Ser. No. 11/009,533) titled “Methods for Generating Immunity to Antigen,” filed Dec. 10, 2004.
The vaccine (see FIGS. 3A-3D) is based on the attachment of a fragment of a TAA fused to the extracellular domain (ecd) of the potent immunostimulatory signal CD40 ligand (CD40L). The TAA/ecdCD40L fusion protein vaccine can be administered either as a TAA/ecdCD40L protein, or as an expression vector encoding the TAA/ecdCD40L such as virus including the adenoviral vector: Ad-sig-TAA/ecdCD40L vector, or other viral vectors, or a plasmid DNA expression vector encoding the TAA/ecdCD40L protein (3-13). The vaccine can be also administered as an Ad-sig-TAA/ecdCD40L vector prime followed in 7 and 21 days with sc injections of the TAA/ecdCD40L protein vaccine. This vaccine platform was developed by the Applicant's laboratory (3-13) to overcome the following problems: weak immunogenicity of the target antigens, qualitative or quantitative defects of CD4 helper T cells, defective response in immunodeficient individuals including the older aged population due to diminished expression of CD40L in activated CD4 helper T cells, and/or low levels of presentation of target antigens on Class I or II MHC in dendritic cells (DCs). The CD40L is important for the expansion of antigen specific CD8 effector T cells and antigen specific B cells in response to vaccination.
Modes of Administration of TAA/ecdCD40L Vaccine.
There are four versions or modes of administration of this vaccine: 1. One in which the TAA/ecdCD40L transcription unit is embedded in a replication incompetent adenoviral vector (Ad-sig-TAA/ecdCD40L); 2. One in which the vector is used as an initial priming injection, followed by two sc injections of the TAA/ecdCD40L protein; 3. One in which the vaccine consists solely of the TAA/ecdCD40L protein; and 4. One in which the TAA/ecdCD40L is inserted into a plasmid DNA expression vector. The TAA is connected through a linker to the aminoterminal end of the ecd of the potent immunostimulatory signal CD40L.
Impact of Attachment of TAA to CD40L.
The attachment of fragments of the TAA to the CD40L accomplishes two things: 1. The binding of the TAA/ecdCD40L protein to the CD40 receptor on the DCs as well as on the B cells and T cells, activating these cells thereby promoting a potent immune response (3, 5, 7); 2. Once the TAA/ecdCD40L protein is engaged on the CD40 receptor of the DC, the entire TAA/ecdCD40L protein is internalized into the DC in a way that allows Class I as well as Class II MHC presentation of the TAA (3, 7).
Activation of DCs by TAA/ecdCD40L Vaccine.
The activated TAA loaded DCs then migrate to the regional lymph nodes (3, 7) where they can activate and induce expansion of the TAA specific CD8+ effector T cells. The antigen specific CD8+ effector T cells become increased in number in the lymph nodes (3, 7), and they then egress from the lymph nodes into the peripheral blood. The antigen specific CD8 effector T cells exit the intravascular compartment and enter into the extra-vascular sites of inflammation or infection (10, 11, and 13). In addition to showing that this vaccine increases the levels of the antigen specific CD8+ effector T cells in the sites of inflammation or infection (12), the Applicant's laboratory has shown that the activation and expansion of the TAA specific B cells by the TAA/ecdCD40L protein increases the levels of the TAA specific antibodies (see FIGS. 3A-3D) including neutralizing antibodies against viral antigens in the serum (10, 11 and 13).